The invention relates to an apparatus for determining an analyte in a fluid, and to a method.
Such an apparatus for determining an analyte in a fluid is known for example in the form of test strips or test systems, as sold inter alia under the trade name “Roche Cardiac Reader”. FIG. 1 shows a known test strip which comprises a plurality of bonded nonwovens and on which a reagent zone and a detection zone are formed. Located in the reagent zone are one or more reagents 1, 2 which react with the analyte 5 to be determined, for example an antigen such as troponin which is present in the fluid. In order to determine the analyte 5, a sample of the fluid is applied to a detachable nonwoven in the reagent one, whereupon the fluid, for example a blood sample, penetrates into the nonwoven material. In the case of blood samples, cellular components are left behind. In the reaction zone formed on the test strip, the analyte 5 to be determined and the one or more reagents 1, 2 react to form at least one reaction product 6. In the illustrated embodiment according to the prior art, a first reagent 1 is a conjugate composed of a first antibody and gold particles. A second reagent 2 is an antibody labelled with biotin 3. The detection zone contains a streptavidin 4 bound to a nonwoven material.
As the analyte 5 to be determined reacts with the first and second reagent 1, 2, the reaction product 6 is produced, which is a sandwich-type compound. By means of capillary, forces, the fluid is transported along the test strip through the different nonwoven sections, wherein the reaction product 6 is bound to the streptavidin 4 in the detection zone by means of the biotin residue 3. Unbound residues 7 are further transported past the detection zone in the test strip to a waste zone. The analyte 5 in the detection zone can then be detected, for example by a quantitative detection based on remission measurements of the absorption values in the detection zone. Other methods for detecting the reaction product are based on electrochemical, other optical or physical methods.
A further embodiment of a known test strip is described for example in the document EP 1 061 369 A2.
However, the known test strips based on nonwoven materials have the disadvantage that a large quantity of the applied sample of fluid is lost within the nonwoven material. It is therefore necessary, for the analysis that is to be carried out, to apply a much greater quantity of sample than would be necessary for the actual determination of the analyte. In order to avoid this disadvantage, there have been proposed apparatuses comprising a three-dimensional structure in which the reagent zone, the reaction zone, the detection zone and optionally the waste zone are arranged one behind the other as regions or sections of the three-dimensional structure and are in fluid connection with one another via a channel structure. Test elements or systems with channel-like structures are thus obtained. In a manner similar to the test strips, in one embodiment, during the determination, a first and a second reagent are mixed with the sample of fluid in the channel-like structure, whereupon the analyte in the fluid reacts with the two reagents to form a reaction product.
As a further zone, a trapping zone may be provided in the three-dimensional structure. This trapping zone is a section of the three-dimensional structure in which for example the second reagent is immobilised, so that a complex composed of the analyte to be determined and the first reagent binds thereto, the complex being formed in a reaction beforehand and thus being a reaction product. The second reagent formed as an antibody can be immobilised directly on a surface in the region of the trapping zone of the three-dimensional structure. Alternatively, an antibody labelled for example with biotin or in an equivalent manner can also be immobilised on the section of the trapping zone which is coated with a suitable receptor. The section of the three-dimensional structure formed as the trapping zone may be a flat surface or a three-dimensional spatial structure. Examples of a three-dimensional trapping zone include porous layers, for example made from a nonwoven, or hydrophilic polymers, for example gels.
The three-dimensional structure for forming the combination of different zones for the test systems may be formed for example in microtitre plates. The three-dimensional structure may preferably be designed as a microfluidic structure. Such structures can be formed from various materials using different production methods, for example by means of laser structure machining or injection moulding techniques. One feature common to the various embodiments is the fact that the sample of fluid containing the analyte to be determined has to be brought into contact with one or more reagents which react with the analyte. For this purpose, it is provided for example in the document WO 98/43739 that one or more reagents are arranged in the region of a surface section of a capillary region of a three-dimensional structure, through which the sample of fluid flows. In this way, in the known test apparatus, a target ligand or a conjugate is immobilised on the surface sections.
On the other hand, in three-dimensional structures into which one or more fluids are introduced, it is known to provide mixing zones for mixing different fluids with one another or for mixing one or more fluids with dry substances. The conditions and thus the mixing processes differ specifically depending on the dimensions of the three-dimensional structure. There are exceptions in the case of microstructures or microfluidic structures, since conditions associated with a low Reynolds number prevail (cf. K Suk et al.: “Micro magnetic stir-bar mixer integrated with parylene microfluidic channels”, Lab Chip 2004, 4, pages 608-613). It has been proposed to use magnetic stirrers to stir fluids in microfluidic structures. In this case, a stirring element is arranged in the microfluidic structure and is driven by means of an external magnetic field. A mixing device based on a similar principle is disclosed in the document U.S. Pat. No. 5,028,142, which can be used inter alia for immunological agglutination assays.
The document U.S. Pat. No. 5,222,808 proposes an apparatus which can be used to analyse liquid biological samples. In said document, the fluid transport in the apparatus is based on capillary farces. In a mixing chamber of the apparatus, reagents which are present in the mixing chamber can be mixed with the sample with the aid of magnetic particles. The analysis of the sample can take place in the mixing chamber itself or in a downstream detection zone by means of optical methods.